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| Author(s): |
Lívia Câmara de Carvalho Galvão
Total Authors: 1
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| Document type: | Doctoral Thesis |
| Press: | Piracicaba, SP. |
| Institution: | Universidade Estadual de Campinas (UNICAMP). Faculdade de Odontologia de Piracicaba |
| Defense date: | 2016-02-26 |
| Examining board members: |
Pedro Luiz Rosalen;
Marcia Pinto Alves Mayer;
Maria Regina Lorenzetti Simionato;
Sonia Maria Fernandes Fitts;
Bruno Bueno Silva
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| Advisor: | Gilson Cesar Nobre Franco; Pedro Luiz Rosalen |
| Abstract | |
Streptococcus mutans is abble to emerge as dominant flora in biofilms even during stress conditions and has been studied in dentistry and medicine. The S. mutans Clp proteolytic system (Caseinolytic protease ATP-dependent) holds a central role in stress tolerance by controlling cellular homeostasis and stability of regulatory proteins. Besides, it was shown that Spx is substrate of Clp what justified a detailed phenotypic characterization of Spx, a global regulator able to regulate transcription of well-known genes related to oxidative stress and others hypothetical genes. Thus, the goal of this study was to construct and phenotypically characterize, in vitro and in vivo, S. mutans knockout mutants of hypothetical genes that showed altered expression in the ?spxA1 and ?spxA1/sxpA2 strains. The mutants of the following genes, regulated by Spx and strong candidates to be related with survival of S. mutans in stress conditions were constructed: smu143c smu144c, smu247, smu248, smu540, smu569, smu570, smu929, smu1296, smu1497 and smu1685. RT-qPCR was used to evaluate the responses of Spx-regulated genes during oxidative stress in the parental and ?spx strains. The mutant strains were characterized by evaluation of its susceptibility, survival and growth under oxidative and acid stresses conditions, as well as in presence of iron and other metals. In addition, their ability to form biofilm in presence of sucrose was evaluated. These genes were also assessed through in vitro transcription assay to confirm their direct regulation by Spx. Selected strains that showed phenotypes associated with oxidative stress management were also evaluated for their ability to colonize using an animal model subjected to high cariogenic challenges. Besides, Spx mutant strains were evaluated for their ability to cause dental caries using an animal model and their regulatory role as competency activator and mutacin, biofilm and glucan sinthesizer. Moreover, mutant for gene smu1784c, also regulated by Spx, was characterized to find phenotypes that justified its relation with eep in Enterococcus faecalis or with oxidative stress in S. mutans. The detailed phenotipic characterization of novel Spx regulated genes lead to their involvment with oxidative stress in S. mutans. Furthermore, these genes and Spx were associated with iron homeostoasis. The role of Spx in caries pathogenesis was confirmed once spxA1 and spxA2 genes were able to reduce caries score in an animal model. Also, smu1784c gene, regulated by Spx, showed involvement with acid and oxidative stresses responses, and virulence of S. mutans once the mutant strain of this gene showed attenuated behavior in Galleria mellonella model. The mutant ?smu1784c was also able to reduce biochemical parameters related with biofilm formation. Therefore, these findings support the regulatory role of Spx enabling the identification of new therapeutic targets usefull for prevention and treatment of S. mutans related diseases (AU) | |
| FAPESP's process: | 12/02278-3 - Construction and phenotypic characterization of Streptococcus mutans mutants strains of genes related to virulence |
| Grantee: | Lívia Câmara de Carvalho Galvão |
| Support Opportunities: | Scholarships in Brazil - Doctorate |